Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
In the yeast Saccharomyces cerevisiae, the Mec1 and Rad53 proteins are involved in the G1, S and G2 cell cycle checkpoint pathways (reviewed by Elledge, 1996). In the presence of DNA damage or a DNA replication block(s), these proteins are required to arrest or slow cell cycle progression. At the same time, they induce transcription of the genes encoding ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in dNTP synthesis that is necessary for replication and repair (reviewed by Reichard, 1988). In addition to their involvement in checkpoint pathways, Mec1 and Rad53 are both essential for cell growth (Zheng et al., 1993; Kato and Ogawa, 1994). This property distinguishes them from most other checkpoint genes, which are dispensable for growth.
Two observations suggest that Rad53 acts after Mec1 for both checkpoint and essential functions. First, the phosphorylation of Rad53 in response to DNA damage is absent in a mec1 mutant (Sanchez et al., 1996; Sun et al., 1996). Second, overproduction of Rad53 suppresses mec1 lethality and partially suppresses its hydroxyurea sensitivity (Sanchez et al., 1996). Since both proteins function as signal transducers in a common checkpoint pathway, they may play a similar role in the regulation of mitotic cell growth. However, the exact nature of their essential functions is not known and whether their checkpoint and cell growth functions overlap is still an open question.
Mec1 is a member of the PI3-kinase family, composed of proteins that have PI kinase or protein kinase activity (reviewed by Zakian, 1995; Shiloh, 1997). Interestingly, ATM and Atm, homologs of Mec1 in human and mice respectively, also play dual roles in cell growth and cell cycle checkpoint function (reviewed by Friedberg, 1995; Shiloh, 1997). Mutations in the ATM gene result in a recessive autosomal disease, ataxia telangiectasia (AT), a multi-system disorder associated with a high risk of cancer. Mitotic cells from AT patients grow poorly, senesce prematurely and exhibit a higher nutrient requirement in vitro. In addition, these cells are checkpoint deficient since they are sensitive to ionizing radiation and display radioresistant DNA synthesis. The phenotype of Atm-deficient mice mimics that of AT patients, and fibroblasts from these mice exhibit growth defects and radioresistant DNA synthesis similar to that observed in AT cells (Barlow et al., 1996; Xu et al., 1996; Xu and Baltimore, 1996; Elson et al., 1996). The growth defect of the Atm-deficient cells correlates with a failure to enter S phase efficiently and can be suppressed by deletion of either the p53 or p21 gene (Xu and Baltimore, 1996; Westphal et al., 1997; Wang et al., 1997b). The molecular basis for the cell growth functions of ATM and Atm is not clear. Given the conservation between Mec1 and these two proteins, an investigation of the role of Mec1 in mitotic growth may shed light on the cell growth functions of ATM and Atm.